Arteries and veins are anatomically distinct within the adult circulatory system, and it was previously thought that the differentiated identities of arterial and venous endothelial cells arose in response to hemodynamic forces such as blood pressure and the direction of blood flow. However, recent evidence suggests that the specification/differentiation of arteries and veins is governed by genetic mechanisms before the active onset of circulation. This event occurs before remodeling of blood vessels, and impaired specification/differentiation of arteries and veins leads to failure to remodel blood vessels. Although it has been shown that several signaling systems such as the VEGF, Notch, and ephrin signaling pathways are involved in this process, molecular mechanisms of how transcription factors function to regulate expression of such genes remain to be elucidated. We have previously shown that the two genes encoding closely related Fox transcription factors, Foxc1 and Foxc2, are expressed in overlapping populations of cells contributing to the endothelial and mesenchymal cells of the blood vessels. Embryos lacking either Foxc1 or Foxc2, and most compound heterozygotes, die pre or perinatally with similar abnormal phenotypes, including defects in the cardiovascular system. Compound Foxc1; Foxc2 homozygotes die earlier and with much more severe defects than single homozygotes alone. Most importantly, compound homozygotes have arteriovenous malformations and the failure of blood vessels to remodel, and in the endothelial cells of compound homozygotes Notch signaling genes and ephrinB2 are downregulated. These data lead to the central hypothesis that Foxc1 and Foxc2 play dose-dependent, interactive roles in the process of arterial-venous cell fate determination/differentiation. One of the goals of this grant is to test whether Foxc proteins act downstream of VEGF signaling to regulate arterial-venous identity (Aim 1). The proposed hypothesis will be tested by (a) analyzing conditional compound mutants of Foxc1 and Foxc2 in endothelial cells crossed with Tie2-Cre mice and (b) rescue experiments in which Tie2-Foxc transgenic mice are crossed with compound Foxcl; Foxc2 mutants (Aims 2). Finally, direct target gene(s) regulated by Foxc1 and Foxc2 in the process of specification/differentiation of arteries and veins will be identified (Aim 3). Elucidating the molecular mechanisms of how Foxcl/c2 function during vascular development will significantly contribute to our knowledge of how genes cooperate to control mammalian cardiovascular development and will lead to a better understanding of human congenital defects.